The goal of this proposal is to adapt recombinant adeno-associated virus (rAAV) vectors for prevention and possibly treatment of persistent hepatitis C virus (HCV) infection in humans. These vectors may be ideally suited for vaccination against HCV because they efficiently transduce muscle and provide potent priming of cellular and humoral immune responses in primates; rAAV vectors also efficiently deliver genes to the liver so they may facilitate targeting or restoration of memory T cell responses at the site of chronic HCV replication. Individuals who spontaneously resolve acute HCV infection rarely develop persistent viremia if reexposed to the virus. We will attempt to replicate protective immune responses by vaccination with rAAV vectors. This includes priming of CD4+ and CD8+ T cell responses that are thought to be essential for protection. Our preliminary studies demonstrate that antibody-mediated depletion of CD4+ or CD8+ T cells in immune chimpanzees prolongs the course of viremia upon rechallenge. Animals lacking memory CD4+ T cells failed to clear the infection through one year of follow-up. Whether neutralizing antibodies contribute to protective immunity is not known, but it is reasonable to postulate that they work in concert with T cells to tip the balance in favor of spontaneous resolution of HCV infection. Neutralization of HCV by serum antibodies from naturally infected or vaccinated individuals has been difficult to study because infectious virus cannot yet be propagated in cell culture. In this project we will use recently developed lentiviral pseudotypes that contain HCV envelope glycoproteins to measure the strength and breadth of neutralizing antibodies primed by rAAV vaccines in mice and non-human primates. These studies, combined with approaches to understand and improve antigen presentation for T cell priming in the liver, should form a foundation for studies in infected chimpanzees and ultimately humans. This proposal has four specific aims: 1. Determine if dendritic cells acquire HCV antigens from rAAV transduced hepatocytes and myocytes for priming of CD8+ T cells, a process known as cross-presentation. 2. Explore strategies for enhanced cross-presentation of HCV antigens. This includes (i) increased dendritic cell migration/maturation in tissues and (ii) conditional apoptosis of rAAV transduced cells to promote antigen uptake by dendritic cells. We anticipate that titers of HCV-specific neutralizing antibodies will be enhanced by these approaches, and will facilitate priming of memory T cells that reside in the liver for long periods of time. 3. Develop a novel rAAV challenge model to determine if liver-resident memory T cells provide accelerated elimination of hepatocytes expressing HCV epitopes. 4. Determine if HCV antigens produced in the muscle of rhesus macaques elicit strong neutralizing antibody and T cell responses, and whether delivery of the vectors to the primate liver leads to intrahepatic targeting of virus-specific T cells.